Histological specimen treatment apparatus and method

ABSTRACT

Method and apparatus for processing tissue specimens against decomposition, putrefaction and autolysis which use a simple three step procedure in a single vessel or container. First, the specimens are saturated with a solvent mixture of a ketone and a hydrocarbon, e.g., an Acetone/Hexane or an Acetone/Xylene mixture, to dissolve lipids and other cellular solutes. The container is then flooded with melted Paraffin. In a last step, the solvent mixture is vaporized and evacuated from the container, allowing the melted Paraffin to replace the vaporized solvent and impregnate the specimens. Raw, i.e., non-processed and non-burred specimens up to 5 mm thick can be processed in about 60 minutes. A solvent regenerator distills the evacuated solvent; and converts vent waste gases into carbon dioxide and water through a thermocatalytic oxidizer.

FIELD OF THE INVENTION

The present invention relates to techniques and instrumentation for thepreservation of tissue specimens. More specifically, the inventionteaches the rapid formation of a matrix by the aggregation of a Paraffinmedium that impregnates and protects the original tissue structure andprevents decomposition, putrefaction, and autolysis.

BACKGROUND

Many of the prior art methods of preparing tissue specimens forhistology use incubation in separate solutions of phosphate-buffered 10%formalin for fixation, a series of increasing concentrations of ethanol,and/or isopropanol for dehydration, and xylene for clearing tissue ofdehydration agent, prior to impregnation. Because of the time requiredfor this process, usually 8 hours or longer, it is customary to completethese separate steps—fixation, dehydration, clearing, andimpregnation—overnight in automated mechanical instruments designed forthose tasks.

The most rapid tissue preservation methods of the prior art rely onmicrowave processing which does not maintain the non-aqueous characterof the reagents that is required in order to prevent burning of thetissue specimens. Moreover, prior fixation processes are plagued byirreversible damage (e.g., hydrolysis of a phosphodiester bond and/ordeamidation) to the structure of nucleic acids (e.g., DNA, andespecially RNA) that limits the application of genetic techniques fordiagnosis and research. Consequently, most DNA and certainly RNAanalysis require special precautions with the handling of material, suchas immediate freezing of fresh tissues to prevent degradation that canimpair retrospective genetic analysis.

Most microwave reagents have poor diffusivity and will not penetrate thespecimen properly without pretreatment. Specimens must be sectioned toabout 1.5 mm or less, and most preferably to about 1 mm or less, for theprocess to work. Specimens also require treatment for 30 minutes or morein a pre-processing solution to harden the specimen, so that it can besectioned to the required thickness.

In some prior processing devices, when the reagents are evacuated afterreacting with the specimens, scale, slime and deposits are formed. Scaleforms where it can be tolerated least-on heat transfer surfaces. It isin this location that the conditions necessary to cause theprecipitation of salts are found. At areas of heat transfer and low flowrates, there is a significant increase in the dissolved solidsconcentration. There is also a localized temperature rise. Thecrystallization of scale on these surfaces is a slow process. Thispromotes the formation of a fairly well defined crystal growth,especially considering the varying composition present in the usedfixative. Slow, in-place crystal growth forms a hard, dense, glassy andhighly insulating material that is deposited on the evacuation valvesand associated conduit surfaces. Some forms of scale are so tenaciousthat they resist any type of removal, mechanical or chemical. Scalesforming on the moving parts of valves stick together and requirereplacement of the valves and valve motors.

There are also problems associated with impeded work flow in thepathology laboratory necessitated by the requisite batch processing ofspecimens, the safety concerns that attend having instruments operatingovernight, the risk of possible instrument failures, the need to monitorthe instruments, and the waste of using large volumes of reagents forsuch processing when automated.

Expensive measures are required to prevent exposure of laboratorypersonnel to noxious fumes and toxic substances associated with thereagents used in this process. Also, the large volumes of reagent wasteand Paraffin debris produced by the conventional methodology willpollute the environment if not properly disposed.

Prior art processing of tissue is accomplished in a plurality ofreactors requiring manual or robotic handling and transfer of thespecimens.

Most significantly, the prior art fails to teach an efficient solvent toremove cellular solutes in a single step.

A typical tissue cell contains: 19.44% ether-extractable material(lipids), 55.13% moisture 18.62% protein, and 5.43% ash. The objectiveis to remove the lipids and free water and replace them with apreserving compound such as Paraffin. Fixation is the first step inpreparing cell and tissue specimens for use in a wide range of clinicaland analytical testing. A good fixative should harden cell and tissuecomponents. The chemical process of tissue modification by a fixative isgradual and complex, involving penetration into the tissue and a varietyof chemical reactions. Formaldehyde fixatives (formalin) are veryreactive electrophilic reagents that fix tissue by covalent bonding toreactive functional groups present in tissue. Neutral Buffered Formalin(NBF) is the most widely used fixative for preserving cell and tissuespecimens. It is relatively inexpensive, simple to use, and providesconsistent results. NBF developed by the US Armed Forces Institute ofPathology is the exclusive aqueous fixative used in automated tissueprocessors. The formulation is a 1:10 v/v dilution of 37% w/vformaldehyde in a phosphate buffer.

However, when tissue specimens are processed at an elevated temperature(35-50 degrees C.) for a long period of time, e.g., 2 to 12 hours, solidmaterials tend to accumulate in the processor. Frequent maintenance isrequired to prevent instrument failure. Several processing devicecomponents can be affected by NBF. They include reservoir connections,rotary valve, retort sensors, overflow sensors, solenoid valves,pressure/vacuum regulator manifolds, process pumps and associatedplumbing.

REVIEW OF THE PRIOR ART

US Patent Application Publication No. 20050090017 describes a processfor simplified tissue processing; the reagent must be water free to workand not burn the tissue specimens. The mixture contains 25% to 60% pervolume acetone, 30% to 55% per volume 2-propanol, and 20% per volumemineral oil. Glacial acetic acid is added at 0.5% per volume and 1% pervolume Dimethyl Sulfoxide (DMSO), (non-aqueous admixture). An example isfor tissue 1.0 to 2.0 mm, preferably about 1.5 mm. The application omitsthe required preprocessing of the specimen. The process used aeration toagitate the mixture. Total processing time is 120 minutes. In thedisclosure, the applicant fails to normalize the relative concentrationsof the non-aqueous blend. The mixture is expressed in volume percentthat usually varies with temperature. There is no correction providedfor the non-linear blending. When, as expected, Acetic acid catalyzesthe esterification (Acid-catalyzed nucleophilic addition) of 2—Propanolyielding an ester and water as equilibrium products, the admixture, onceblended, is no longer non-aqueous. An Hydronium ion is present, andcondenses Acetone to Diacetone alcohol. The condensation is enhanced bythe heater microwaves. Moreover, aeration increases the waterconcentration.

Recent U.S. Pat. Nos. 6,207,408; 6,586,713; and 6,793,890 addressed to asimilar process also fail to deal with condensation products.

U.S. Pat. Nos. 3,892,197; 4,141,312; and 5,049,510 exemplify theconventional method of fixing tissues by incubation in separatesolutions of phosphate buffered 10% Formaldehyde, dehydration byincreasing concentrations of Ethanol and/Isopropanol, and clearing thetissues of dehydration agents prior to impregnation with Xylene. Becauseof the time required to perform the several steps, typically eighthours, automated devices are run overnight.

Currently, the most advanced automatic tissue processor of this type ismarketed under the brand name TISSUE-TEK by Sakura Finetek of Torrance,Calif. The SAKURA TISSUE-TEK vacuum infiltration processor requires morethan eight hours for completion of processing. Baskets holding thecassettes are placed in a retort in which tissue is processed. Inaddition, 14 stations supply solutions of various compositions to theretort. User-programmable software controls this automated process. Arotary valve regulates the movement of solutions between the retort andthe various stations, applying pressure or vacuums to the retort whenthe valve is open causes solution to be pumped out of or pumped into theretort, respectively. Upon completion of a processing run, theinstrument automatically prompts the user for a cleaning cycle; thisrequirement can be overridden only if no Paraffin is used.

Typically such conventional methodology demands sending tissue specimensfrom the operating room, medical office or other sites, to a pathologylaboratory sometime during the working day; overnight batch processingof the specimens, so that a tissue specimen suitable for blocking andsectioning is only available on the morning of the next day; andrendering a diagnosis by a pathologist based on microscopic examinationof sections prepared from a blocked and sectioned specimen later on thatnext day. This requires almost 24 hours between receipt of the specimenand delivery of the pathologist's report. Although a shortened versionof the conventional method is presently practiced, it is feasible onlyfor small biopsies. These biopsies need to be fixed for at least about30 minutes before initiating the processing cycle. The instrumentprocessing cycle can be programmed to last a minimum of 70 minutes, butis preferably 2 to 2½ hours. The SAKURA FINETEK microwave systemrequires samples to be pre-processed in a pre-processing solution(Sakura P/N 7115) for 30 minutes prior to sectioning. Tissue sectionsmust be less than 2.5 mm before microwave processing. The system maytake 3-hours or more. Specimens may have to be reprocessed due to sampleburning, resulting from poor microwave control, accumulation ofextracted cellular solute in the reagent, and/or additional problemsinherent to microwave processing. Microwave processing reagents aretoxic, corrosive, chemically unstable, form reactive azeotropes, and areinsoluble in Paraffin and thus cross-contamination of microwavereagents.

Boon et al. (Eur. J. Morphol. 33:349-358, 1995) use an isopropanolsolution and a Paraffin wax solution in two separate reaction chambers,each subject to vacuum and microwave heating, to process tissuespecimens for histology. That system requires that tissue specimens befixed prior to processing and uses a turntable to distribute microwaveenergy. In addition, the glass container holding the tissue specimensalso adsorbs microwave energy and transfers heat to the solutiontherein.

Milestone (WO 98/05938) provides another alternative for tissueprocessing in at least three steps: fixing the tissue specimen,simultaneously dehydrating and clearing with dehydrating agent and anessentially lipophilic agent, and impregnating the tissue specimen.Microwave heating is used in the first two steps and elevated pressureis used during the dehydrating/clearing step. The tissue specimen isdried by heating and reduced pressure, and then it is impregnated undervacuum or, alternatively, by applying a cycle of moderate pressure andmoderate vacuum.

Therefore, in summary, with current microwave techniques, samples haveto be pre-processed in a pre-processing solution, and cut to 1.5 mm orless in thickness, loaded into a loading station and processed usingmicrowaves through a series of chemical reagents. The process may take3-hours or more and samples may have to be reprocessed because of sampleburring resulting from poor microwave control or the presence of waterin the reagent.

Considering that the objective is to remove cellular solutes thatinterfere with Paraffin preservation, and to have a specimen retain itslifelike characteristics, it is critical to keep in mind that theextraction of cellular solute is a function of the moleculardiffusivity, solubility of the solute in the solvent, and theconcentration gradient in and outside of the specimen. The followingequation can then be used to predict molecular diffusivity at a fixedconcentration of solvent:

D _((solvent→Tissue)) =kT/6πμr  (Equation No. 1)

where:μ=Viscosity in posesr=radius of diffusing moleculek=experimental diffusion constantT=temperature in degrees K

The equation suggests that only small molecules with low viscosity couldeffectively penetrate tissue, and effectively extract cellular solutesat temperatures less than 100° C. Larger molecules like mineral oils asused in U.S. Pat. Nos. 6,793,890, 6,207,408, and 6,586,713 do not havethe proper effect on extraction of cellular solute. In these patents,mineral oil accumulates on the surface of the specimen, causing poorParaffin adhesion; Oil also contaminates the Paraffin storage vesselresults in poor sectioning over the life of the Paraffin. The Paraffinmust be replaced at regular intervals.

Ideally, the extraction solvent should have the followingcharacteristics:

-   1. It should have a high capacity for the species being separated    into it, namely, water and water soluble cellular components that    interfere with Paraffin impregnation.-   2. It should be selective, dissolving one or more of the components    being separated to a large extent while not dissolving the other    components to any large extent.-   3. It should be chemically stable.-   4. It should be regenerable, so that extracted species can be    separated from it readily and it can be reused again and again.-   5. It should be inexpensive to keep cost of maintaining solvent    inventory and replacing lost solvent low.-   6. It should be nontoxic and noncorrosive.-   7. It should have a low viscosity.-   8. It should not form so stable an emulsion that the phase cannot be    separated adequately.-   9. It should allow formation of immiscible liquid phases.

SUMMARY

In a single pressure and temperature controllable reaction container, arapid and automatic extraction of cellular solutes that interferes withthe impregnation of liquid Paraffin into tissue specimens is combinedwith the impregnation of the evacuated space with Paraffin. Inparticular, some embodiments of the invention optimize reactionparameters such as: temperature, pressure, solvent flow rate, reactioncontainer space velocity, and solvent properties, so that specimens arefixed, dehydrated, cleared, and impregnated to produce a Paraffinizedtissue section in one hour or less.

In some embodiments the solvent is recovered and its volatile componentsare run through a thermocatalytic oxidizing system that convert them tocarbon dioxide and water.

In some embodiments the reliance on a mixture of a ketone and a lowmolecular weight hydrocarbon is based on the following considerations:

1) The inventor concluded that ketones, existing in the liquid state canbe used as a primary extraction solvent based upon the above statedequation. They are soluble in cellular solutes and are present in lowconcentrations or are absent in biological systems. Acetone is aneffective solvent for small specimens, less than 1.5 mm. Being polar,Acetone will partition itself into the aqueous phase. It has problemsextracting large molecular weight lipids that have hydrophobic groups.Paraffin cannot be directly blended with Acetone. It is also insolublein Acetone.

2) When ketones are mixed with low molecular weight hydrocarbons, asuitable extraction solvent is produced. One such mixture is Acetone andHexane or mixed Hexanes (A/H). The mixture is excellent for extractingwater, lipids, and other cellular solutes that will interfere withParaffin impregnation. The mixture also has some unique properties. Thesolvent Dimethyl Sulfoxide (DMSO) is insoluble in Hexane, and Paraffinis insoluble in Acetone. However, an Acetone-Hexane mixture willdissolve both DMSO and Paraffin. That type of dissolving compoundmixture has both polar and non-polar regions within the mixture. A blendof about 60% per weight Acetone and 40% per weight Hexane forms anazeotropic mixture with a boiling point of 49.8° C. The azeotrope can beeasily separated from the cellular solutes and regenerated foradditional use. Accordingly, the preferred solvent is an azeotropic,non-aqueous dehydrant, that is a solvent mixture that maintains a lowboiling point and a low viscosity when combined with the solutes foundin tissues. Preferably, the dissolving compound mixture should exhibit aviscosity of no more than 0.35 centipoise at 60° C.

3) The A/H compound is an ideal solvent for tissue preservation. It canremove all necessary cellular components that interfere with Paraffinimpregnation in a short time, 10 to 30 minutes. The A/H solvent willsaturate a tissue specimen, replace the extractable cellular solutes,and can be removed under normal operation conditions as taught in thepresent invention. It provides a vehicle for transporting Paraffin intothe specimen resulting is very short impregnation times.

4) The throughput-limiting factor in tissue processing is the rate ofParaffin impregnation. The experimental value for diffusivity ofParaffin into breast tissue is 5.0×10⁻⁶ cm²/sec, at 60° C. and −27inches Hg. Thus, for a 1.0 mm specimen the impregnation time is greaterthan 60 minutes. By using an A/H solvent and adding 6% per weight ofParaffin, the impregnation time for a 5.0 mm specimen is only 30minutes. The blending of the Paraffin with the solvent reduces theviscosity of the Paraffin and allows for faster penetration.

A zeotropic solvent comprising a mixture of Acetone and Xylene or mixedXylenes (A/X) may also be advantageously used.

The present embodiments may include a buffer to stabilize the extractionsolvent to near neutral conditions. Tris-(hydroxymethyl)aminomethane isan excellent buffer for this application, but any compound capable ofbuffering the solvent at near neutral pH and which is soluble in theextraction solvent is applicable. Other chemicals may be added to theA/H or A/X solvent to increase solvent viscosity. Examples are alcohols,ketones, aldehydes, solvents, and hydrocarbons, e.g. DMSO and2-propanol. DMSO is an additional extraction solvent that can added tothe A/H or A/X solvent. DMSO assists in the extraction of large ringcompounds and free nucleotides, e.g., lithocholic acid, deoxycholicacid, cholesterol, and other compounds that interfere with Paraffinimpregnation. Alcohols may be added to slow the extraction process andprevent cellular membranes from rupturing.

The present embodiments do not use microwaves. Specimen processing canbe completed in approximately one hour. There is little or nosignificant pre-processing of specimens prior to solvent extraction.Specimens as large as 5 mm can be readily processed.

In some embodiments homogeneous temperature profiles are seen betweenspecimens and solvent. The solvent extraction process can take place ina single pressurized reactor at pressures of between −29.9 inches Hg to50 psig, extraction temperatures between 30° C. and 100° C., and typicalspace velocities between 30 and 150 seconds.

In the disclosed embodiments the presence of water in the solvent doesnot affect the reactor's temperature profile or the specimen's lifelikecharacteristics as can happen with microwave processing. Excess waterfrom cellular solute extract and other cellular molecules can be furtherseparated during solvent regeneration.

In some embodiments the process does not use microwaves to heat thespecimen and chemical reagents. Therefore, a homogeneous temperaturethroughout the reaction container can be readily maintained. In someembodiments the reaction takes place in a single reaction container, atelevated pressures up to about 3.3 bars (50 psig), temperatures 30° C.to 100° C., and a space velocity between 30 to 150 seconds. In someembodiments the solvent may include Paraffin. In some embodimentsParaffin need not be separated from the solvent in a separate vessel,and there are no robotic or manual transfers of specimens. In someembodiments the Paraffin concentration in the solvent can to increaseover the life of the solvent without affecting the lifelikecharacteristics of the specimens. In some embodiments thesolvent/Paraffin mixture can also be used to lubricate pumps used in theprocessor. The solvent blend can be chemically stable.

In some embodiments specimens are saturated with this dissolvingcompound. In some embodiments Paraffin impregnation of the specimensdoes not require vacuum drying or transfer to another vessel prior toParaffin impregnation as with microwave processing. Specimen and solventtemperatures are homogenous within the reaction container onceequilibrium has been reached. In microwave systems, there are differentheat transfer rates; the specimens may contain more water than thereagent or vise versa; and different heating rates are used.Consequently, inconsistent results are obtained. Many of the presentembodiments provide consistent, lifelike results, faster, withoutpre-processing requirement, with less cutting (Often, specimens up to 5mm thick can be processed), without solvent cross-contaminationproblems, without having to burr specimens, without water contaminationproblems, and in a single reaction container without the safety problemsassociated with microwave systems.

Some embodiments provide a tissue flow processing method whichcomprises: providing a pressure and temperature controllable reactioncontainer; placing at least one fresh tissue specimen in said container;treating said specimen in said container to remove solutes and replacesaid solutes with Paraffin, said step of treating consisting essentiallyof: introducing a temperature and pressure conditioned dissolvingcompound into said container; allowing time for said compound topenetrate said specimen and to dilute solutes therein; flooding saidcontainer with liquid Paraffin; vaporizing said compound and allowing aperiod for said Paraffin to impregnate said specimens; evacuating saidcompound and diluted solutes from said container; and, reducing thetemperature in said container below the melting point of Paraffin.

In some embodiments, the dissolving compound comprises a zeotropic or anazeotropic dehydrant mixture.

In some embodiments the dissolving compound comprises a solvent takenfrom a group consisting essentially of ketones, esters, alcohols,aldehydes, cyclic compounds, ethers, cyclic ethers, aromatics, lowmolecular weight hydrocarbons, and a mixture of low molecular weighthydrocarbons.

In some embodiments the dissolving compound consists of a mixture takenfrom a group consisting essentially of:

a mixture of about 60% per weight Acetone and about 40% per weight atleast one Hexane;

a mixture of about 46% per weight Acetone, about 25% per weight at leastone Hexane, about 7% per weight DMSO, about 20% per weight 2-Propanoland about 2% per weight Paraffin;

a mixture of about 62% per weight Acetone, about 16% per weight at leastone Hexane, about 4% per weight DMSO, about 12% per weight 2-Propanoland about 6% per weight Paraffin;

a mixture of about 17% per weight Acetone, about 39% per weight at leastone Hexane, about 5% per weight DMSO, about 12% per weight 2-Propanoland about 30% per weight Paraffin;

a mixture of about 46% per weight Acetone, about 25% per weight at leastone Hexane, about 7% per weight DMSO and about 20% per weight2-Propanol;

a mixture of about 53% per weight Acetone, about 26% per weight at leastone Xylene, about 20% per weight Ethanol, and about 1% per weight DMSO;and,

a mixture of about 62% per weight Acetone, about 19% per weight at leastone Xylene, and about 19% per weight Ethanol.

In some embodiments the method further comprises regenerating dissolvingcompound evacuated from said container in a regeneration canister. Insome embodiments the regenerating comprises distilling said evacuatedcompound; and converting vent waste gases into carbon dioxide and waterthrough a thermocatalytic oxidizer. In some embodiments the methodfurther comprises conditioning regenerated dissolving compound forintroduction into said reaction container. In some embodiments theconditioning comprises heating said dissolving compound in solventvessel to a temperature of approximately 60° C.; and establishing apressure of approximately 0.8 bars (12 psig) in said solvent vessel. Insome embodiments the time allowed for the dissolving compound topenetrate the specimens is limited to no more than approximately 30minutes, and said period is limited to no more than approximately 30minutes. In some embodiments the specimens may be taken from a groupconsisting essentially of non-processed and non-burred tissue sections 5mm thick. In some embodiments the penetration time is limited to no morethan approximately 30 minutes, and said period is limited to no morethan approximately 50 minutes; and the specimens comprise non-processedand non-burred tissue sections 10 mm thick. In some embodiments themethod further comprises admitting a limited amount of Paraffin into thecontainer before introducing said the compound.

In some embodiments there is provided a histoprocess which comprisesdissolving and removing cellular solutes in a tissue specimen using asingle dissolving compound; and replacing said solutes with Paraffin;said process being performed in a continuous sequence of steps within asingle vessel. In some embodiments the dissolving compound comprises asolvent taken from a group consisting essentially of ketones, esters,alcohols, aldehydes, cyclic compounds, ethers, cyclic ethers, aromatics,low molecular weight hydrocarbons, and a mixture of low molecular weighthydrocarbons. In some embodiments the dissolving compound comprisesAcetone and Hexane.

In some embodiments there is provided a method for treating histologicalspecimens having a sequence of steps which comprises: saturating saidspecimen with said dissolving compound; flooding said vessel with liquidParaffin; and vaporating said dissolving compound and said solutes toallow said Paraffin to infiltrate spaces vacated by said compound andsolutes.

In some embodiments the steps further comprise: evacuating said mixtureand solutes to a regeneration canister; recuperating said mixture bydistillation; and disposing of vent gases by thermocatalytic oxidation.In some embodiments the process further comprises mixing said compoundin said vessel prior to said dissolving and removing.

In some embodiments there is provided an apparatus for processing tissuespecimens against decomposition, putrefaction and autolysis whichcomprises: a hermetically sealable reaction container; means forcontrolling the flow, temperature and pressure inside said container; asource of dissolving compound connectable to said container; a source ofmelted Paraffin connectable to said container; and means for evacuatinggases from said container; wherein said dissolving compound comprises amixture of a ketone and at least one low molecular weight hydrocarbon.

In some embodiments that dissolving compound consists of a mixture takenfrom a group consisting essentially of:

a mixture of about 60% per weight Acetone and about 40% per weight atleast one Hexane;

a mixture of about 46% per weight Acetone, about 25% per weight at leastone Hexane, about 7% per weight DMSO, about 20% per weight 2-Propanoland about 2% per weight Paraffin;

a mixture of about 62% per weight Acetone, about 16% per weight at leastone Hexane, about 4% per weight DMSO, about 12% per weight 2-Propanoland about 6% per weight Paraffin;

a mixture of about 17% per weight Acetone, about 39% per weight at leastone Hexane, about 5% per weight DMSO, about 12% per weight 2-Propanoland about 30% per weight Paraffin;

a mixture of about 46% per weight Acetone, about 25% per weight at leastone Hexane, about 7% per weight DMSO and about 20% per weight2-Propanol;

a mixture of about 53% per weight Acetone, about 26% per weight at leastone Xylene, about 20% per weight Ethanol, and about 1% per weight DMSO;and,

a mixture of about 62% per weight Acetone, about 19% per weight at leastone Xylene, and about 19% per weight Ethanol.

In some embodiments the apparatus further comprises means forregenerating vaporized dissolving compound evacuated from saidcontainer. In some embodiments the means for regenerating comprise: astill for recuperating said evacuated dissolving compound; and athermocatalytic oxidizer for converting vent waste gases into carbondioxide and water. In some embodiments the dissolving compound in theapparatus comprises a mixture of approximately 17% to 65% per weightAcetone and 16% to 40% per weight Hexane.

In some embodiments there is provided a combination of a tissue specimenwith a preparation for dissolving lipids and other cellular solutes insaid specimen taken from a group consisting essentially of:

a mixture of about 60% per weight Acetone and about 40% per weight atleast one Hexane;

a mixture of about 46% per weight Acetone, about 25% per weight at leastone Hexane, about 7.0% per weight DMSO, about 20% per weight 2-Propanoland about 2.0% per weight Paraffin;

a mixture of about 62% per weight Acetone, about 16% per weight at leastone Hexane, about 4% per weight DMSO, about 12% per weight 2-Propanoland about 6% per weight Paraffin;

a mixture of about 17% per weight Acetone, about 39% per weight at leastone Hexane, about 5% per weight DMSO, about 12% per weight 2-Propanoland about 30% per weight Paraffin;

a mixture of about 46% per weight Acetone, about 25% per weight at leastone Hexane, about 7% per weight DMSO and about 20% per weight2-Propanol;

a mixture of about 53% per weight Acetone, about 26% per weight at leastone Xylene, about 20% per weight Ethanol, and about 1% per weight DMSO;and,

a mixture of about 62% per weight Acetone, about 19% per weight at leastone Xylene, and about 19% per weight Ethanol.

In some embodiments there is provided a process, for removing cellularsolutes from a tissue specimen, which comprises treating said specimenwith a dissolving compound having a viscosity of no more than 0.35centipoises at 60° C. and a boiling point of less than 80° C. at 10torr. In some embodiments the dissolving compound is regenerated aftercombination with the solutes.

In some embodiments the dissolving compound is regenerated aftercombination with the solutes. In some histoprocesses, the dissolvingcompound comprises a lubricant for process pumps and valves. In someembodiments, the Paraffin present in the solvent provides lubricatingproperties for process pumps and valves. In some embodiments, thedissolving compound is vaporizable at a pressure of no more than 10 torrand a temperature of no more than about 80° C.

In some embodiments, the process for removing cellular solutes from atissue specimen comprises treating said specimen with a zeotropicdissolving compound having a viscosity of no more than 1.30 centipoisesat 60° C. and a boiling point of less than about 80° C. at a pressure ofno more than 10 torr. In some embodiments, the dissolving compound has arefractive index of between about 1.350 and about 1,499 at 20° C.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a flow diagram of the tissue preserving process according tothe invention;

FIG. 2 is a diagrammatical illustration of the apparatus to practicesaid process; and

FIG. 3 is a diagrammatical illustration of the solvent regenerationportion of the apparatus.

DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

Referring now to the drawing, there is shown in FIG. 1 the basic stepsof a tissue specimen preserving process 10 according to the invention,and, in FIGS. 2 and 3 the instrumentation 25 that may be used topractice the process.

In a hermetically sealable, and pressure and temperature controllablereaction vessel or container 11 in which one or more tissue specimensare being held, a limited volume of melted Paraffin is admitted 12 froma storage tank 13. The Paraffin has been preferably conditioned 14 byraising its temperature slightly above its melting point, e.g., 60° C.

A compound formulated to dissolve cellular solutes in the specimens isinjected 15 into the container. These solutes include lipids and otherhydrous elements. The compound penetrates the specimens, diluting 16 thesolutes. The container is the flooded with Paraffin which saturates 17the specimens as the dissolving compound is being vaporized andevacuated 18 from the vessel. After removal 19 of excess Paraffin, thereaction vessel is cooled 20 allowing retrieval of the treatedspecimens.

The evacuated dissolving compound carrying the removed solutes isregenerated by distillation 21 and by converting 22 the waste gases intocarbon dioxide and water through a thermocatalytic oxidizer.

The recovered dissolving compound is stored 23 then pressure andtemperature preconditioned 24 just prior to being used again in thereaction vessel.

The processing steps and required instrumentation will be now describedin greater details.

As illustrated in FIG. 2, the key components of the histoprocessinginstrumentation 25 comprise a hermetically sealable, pressure andtemperature regulated reaction container 11 sized to hold a number oftissue samples 26. The reaction container is connected by a network ofconduits and valves to a source of melted Paraffin constituted by aParaffin makeup vessel 27, to a source of dissolving compound in theform of a solvent regenerator 28 drawing from a chemical tank 29, to asolvent pump 30, to a vacuum pump 31, and to an overflow reservoir 32.The solvent pump is designed to operate over a wide range oftemperatures and pressures.

As shown in FIG. 3, the solvent regenerator 28 is connected to a solventdistillation assembly 33 including an accumulator 34, a recirculationpump 35 and a condenser 36. A thermocatalytic oxidizer 37 is used tobreakdown waste gases into water and carbon dioxide. A carbon bed devicecould be substituted for the oxidizer. The waste material extracted formthe used dissolving compound is sent to a waste tank 38. A processheater 39 is provided between the solvent pump and the reaction vessel.A heat exchanger 60 consisting of a 500 watt, explosion-proof bulb ismounted inside the solvent regenerator. A jacket heater 40 surrounds thesolvent regenerator. A similar jacket heater 65 surrounds the reactioncontainer. A series of snubber protected pressure sensors 64, 72 and 73are connected to various areas of the system to regulate pressures.

Valves 41-59, and the pumps 30, 31 and 35 are controlled by aprogrammable electronic unit 41 according to techniques well known inthe industrial process arts.

The process of treating tissue specimen according the inventionpreferred mode runs as follows.

Tissue specimens are loaded into a stainless steel or polymerconstructed basket, and then placed into the reaction container 11. Thecontainer cover is then closed and latched. Proximity sensors detect thelid and the process starts. Valve 49 opens and the vacuum pump 31 isstarted. The pressure of the system is reduced below ambient atmosphericpressure. Valve 49 closes, the vacuum pump is turned off and the systemcomes to pressure equilibrium. If this condition is not achieved withinone minute, a leak is assumed, and the system is shut off.

Preconditioned dissolving compound is transferred from the solventregenerator 28 to the reaction container 11. Valves 43, 44 and 45 areopened and the reactor jacket heater 65 is energized. The solvent pump30 and heater 39 are energized. Hot compound fills the reactioncontainer until a solvent level detector switch 67 is triggered. Thepressure in the reaction container is regulated by opening valve 47 andthe reactor's pressure sensor 64. Valve 45 closes and valve 42 opens.The dissolving compound is recycled and heated in the closed loopconsisting of valves 42, 43, and 44, heater 39, solvent pump 30, and thereaction container. At this time, the solvent pump is used to pump upthe reactor to an operating pressure as high as about 3.3 bars (50 psig)are used.

Specimens are exposed to the dissolving compound for 10 to 30 minutes.During this exposure, cellular solutes are extracted, (e.g., water,lipids, etc.), and replaced with a mixture of liquid Paraffin and thelow molecular hydrocarbons of the compound. Valve 42, 43 and 44, theprocess heater 39, and the solvent pump 30 are turned off. Hotdissolving compound is transferred from the reaction container to thesolvent regenerator. The transfer path includes valves 42, 43, and 44.Valve 47 is opened between the reaction container and the solventregenerator in order to equilibrate pressure. The transfer lasts untilthe container's low level indicator switch 63 is reset. At this time,valve 42 is closed and the compound is recycled and reconditioned aslater explained. The reaction container 11 is next flooded with liquidParaffin as follows. Valve 42 and 49 open, the vacuum pump 31 is turnedon. Liquid Paraffin flows into the container until the level indicatorswitch 67 is triggered. A high level indicator switch 68 in thecontainer acts as a fail-safe device. At this time valve 42 is closed.The saturated specimens are then subjected to a vacuum to extractvolatiles.

The pressure in the container is further reduced to evaporate the alldissolving compound present. The diffusion pump 66 is used to reducepressures to less then 1 torr. Vacuum is applied until pressureequilibrium is achieved, e.g. about 0.9 to 0.99 At. (−27 to −29.91inches Hg), depending upon solvent. Once equilibrium is reached, allvolatile solvent molecules have been removed from the reaction containerand specimens. The process is allowed to continue for an additional 10to 30 minutes depending upon total mass of specimens.

The vacuum system consists of the vacuum pump 31, the diffusion pump 66,and the overflow vessel 32 equipped with a proximity sensor 71, and anisolation valve 49. The overflow vessel acts as an additional fail-safedevice in case of failure of the high level indicator switch 68. The airmakeup valve 50 is provided to dilute gases prior to entering thethermocatalytic oxidizer 37 and to supply cooling gases to the reactioncontainer during cool down cycles. The pressure sensors 64, 72 and 73are connected to the control unit 41 in order to monitor all processesof the instrument. Snubbers are employed to prevent liquid from enteringthe pressure sensors.

In a final step, the Paraffin is returned to the Paraffin makeup vessel27 as follows. Valves 42, 49 and 50 are opened and the vacuum pump 31 isturned off. Cooling air is drawn through valve 50 and routed to thereaction container. Paraffin is gravitated to the Paraffin makeup vessel27. During this time, the specimen temperature drops below the Paraffinmelt point. The lid of the container opens, the specimen tray iswithdrawn and the specimens are extracted and separated.

The following alternate batch solvent blending process may be practicedto prepare a Paraffin-loaded dissolving compound.

Referring to FIG. 2, the reaction container 11 is hermetically sealed. Ameasured volume of Paraffin-free based solvent is drawn from thechemical tank 29 through a coupler 75 and transferred to the solventregenerator 28 where it is heated to 60° C. The jacket heater 40 of thesolvent regenerator and the vacuum pump 31 are energized. A normallyclosed control valve 42 is opened and liquid Paraffin is transferredfrom the Paraffin makeup vessel 27 to the reaction container 11 until aParaffin level detector 62 inside the reaction container is triggered.At this time, the control valve 42 is closed, and the vacuum pump 31 isstopped. Control valves 43, 44, and 45 are opened and solvent istransferred from the solvent regenerator to the reaction container. Whenthe level detection switch 67 in the reaction container is triggered,the solvent pump 30 is stopped. At this point, all control valves arepositioned to create the loop configuration described earlier. Theprocess heater 39 and the solvent pump 30 are energized. The solvent andParaffin are allowed to blend for about ten minutes into the finaldissolving compound. The circuit is reconfigured to transfer the entireblended compound to the solvent regenerator. The reaction container isevacuated. The system is now ready to process specimens. The abovedescribed blending sequences of solvent and Paraffin will be omitted insome of the examples described below.

Solvent Regeneration

Recovery and regeneration of the dissolving compound from processstreams is desirable, both economically and ecologically. The objectiveis to recover, purify, and re-use extraction solvents by isolating andfiltering cellular solutes from the dissolving compound for wastedisposal.

The dissolving compound recovery and regeneration system illustrated inFIG. 3 runs seamlessly with the histoprocess, and does not requirefrequent attention. For illustration purposes, the following process isdescribed in connection with the Acetone-Hexane (A/H) compound. Therecovery and regeneration system is designed to work with many differentsolvent blends, including both azeotropic and zeotropic blends.

Hot dissolving compound loaded with the removed cellular solutesevacuated from the reaction container 11 is returned to the solventregenerator 28. A Wheatstone bridge type of conductivity detector 61associated with the solvent regenerator senses the presence of excesswater and cellular solutes, and compares this measurement with thereference conductivity for the pure dissolving compound. When there is anoticeable difference between the measurement and a reference value, hotcompound is routed to the packed column 58.

The regeneration process goes as follows. A signal is sent from theconductivity detector 61 to the control unit 41 to initiate the process.Valves 45, 47,49 and 57 open, the recirculation pump 35 is energized andthe compound is transferred to the column 58. The process continuesuntil a very low level indicator switch 70 in the solvent regenerator isopened. At this point, the recirculation pump is turned off, and allvalves are closed. The entire dissolving compound has now beentransferred from the solvent regenerator to the column. If vacuumdistillation is required, valves 47, 49, 51, 54, and 55 open. The vacuumpump 31 is energized and the system is pumped down to the requiredvacuum. Heaters inside the column are activated and the internaltemperature is stabilized to 60° C. The condenser 36 and therecirculation pump 35 are energized and the compound is distilled,super-cooled, and collected in the accumulator 34. Valve 57 is partiallyopened to recycle the product. When the compound reaches a levelindicator in the accumulator, valve 54 opens and solvent is transferredto the cooled solvent regenerator 28. The column is optimized bypressure control using valve 55. The control valve 55 provides a bypassfrom the vapor line to the accumulator. When valve 55 is opened, itequalizes the pressure between the vapor line and the accumulator 34.This causes the condenser 36 to become flooded with condensate. Theflooding of the condenser causes the pressure to build up because of thedecrease in the active heat transfer surface available decreasing theoverhead flow from the column 58 and improving the internal reflux ofthe column. Under normal operating conditions, super-cooling thecondensate causes fluid flow from the condenser 36 to the accumulator 34and this flow is sufficient to reduce the vapor pressure in theaccumulator 34, and allow the column 58 to come to equilibrium foroptimized distillation. When the condensing pressure is to be reduced,valve 55 closes, resulting in an increase in the exposed condensersurface area. In order to expose more area, the condensate istransferred to the accumulator. This transfer can occur only if theaccumulator vapor pressure has been sufficiently lowered bycondensation. Therefore, the system speed in this direction is afunction of condensate super-cooling. Increased super-cooling increasessystem response speed.

The process continues until there is no more liquid buildup in theaccumulator. At this point, there is a change in the system pressuresensed by the pressure sensor 73, valve 57 closes and all remainingliquid in the accumulator is transferred to the solvent regenerator. Therecirculation pump 35 turns off, all valves close and the heater in thecolumn is turned off. A sensor in the column measures the bottomtemperature and when that temperature reaches 60° C., valve 56 opens andthe column bottom waste flows into the solvent waste trap 74. A liquidindicator switch is positioned on this trap and opens and closes valve59. The column waste flows into the waste collection vessel 38 though abottle coupler 69. When the liquid indicator senses that no fluidremains in the waste trap, valve 59 closes.

Waste Gas Treatment

Vent waste gas (i.e., dissolving compound waste gas) can be disposed ofby two methods. The first approach is carbon adsorption currently usedin most histoprocessing. Once the carbon is saturated with solvent, itmust be treated prior to disposal. A preferable alternative in thepresent invention users thermocatalytic oxidation. When properlycontrolled, that process results in a 99.999% conversion of the gas tocarbon dioxide and water.

The thermocatalytic oxidizer assembly includes the following elements: athermocatalytic oxidizer 37, flue gas control valve 76, a recycling paththrough valve 77, and a flue gas after-cooler 78. A typicalthermocatalytic oxidizer comprises two distinct sections, namely, apreheating chamber to elevate the gases to proper reaction temperature,and a catalytic oxidation section to oxidize gases into carbon dioxideand water.

Vent and relief valve gases are routed through the vacuum system to thethermocatalytic oxidizer, either by direct or by venturi connections.Inline thermal conductivity detectors monitor correct vent gasconcentration and trigger dilution of these gases with air through valve50, to obtain the correct reaction mixture. The mixture is thenpreheated about 500° F. to initiate the reaction. Gases then passthrough a heated catalyst bed, where they react to form water vapor,carbon dioxide, and inert gases. Temperature indicators are used tostabilize the reaction temperature. Flue gas detectors are used toanalyze the gas for complete oxidation, and, through the control unit,determine the correct amount of recycle gas, and the proper degree ofaperture for the flue gas control valve in order to optimize thereaction.

Temperature and Pressure Preconditioning of the Dissolving Compound

The cellular solute extracting mixture is loaded into the chemical tank29. A bottle coupler 75 provides a connection for solvent transfer andventing. Valves 46, 44 and 43, are open and the solvent pump 30transfers mixture into the solvent regenerator 28. Valves 47 and 49 areopened to provide a path to the thermocatalytic oxidizer 37. Once themixture is transferred from the chemical tank to the solventregenerator, valves 46 and 47 close. The transfer is monitored by levelcontrol switches. Valve 45 is opened and the mixture is heated in theregenerator loop, using the process heater 39 and the regenerator jacketheater 40.

Vent gases are delivered to the thermocatalytic oxidizer 37 foroxidation to carbon dioxide and water as follows. Valve 50 is opened,the vacuum pump 31 is energized. Waste gases are delivered to theoxidizer. Once the inline thermal conductivity detector detects roomconditions, valve 49 and 50 close and the vacuum pump is turned off. Themixture is heated to the desired temperature and pressure, e.g. 60° C.and about 0.8 bars (12 psig). It should be noted that the solventregenerator operating conditions are flexible, and that it can heatedfrom 20 to 100° C. and pressurized to from 1 to about 3.5 At. Inaddition, the variable flow and pressure solvent pump will operateequally over a wide range of temperatures and pressures. A flow orificemay be placed on the discharge to insure proper pump lubrication. Also,it is worth noting that the Paraffin present in the solvent alsoprovides lubricating properties for pumps and valves.

Example 1

TABLE 1.0 Composition of the dissolving compound ComponentWeight-percent Acetone 45.8 Hexane 25.4 DMSO 7.0 2-propanol 19.8Paraffin 2.0

TABLE 1.1 Typical processing times and specimen type. ProcessingReference Color code Time Impregnation Tissue No. Code (minutes) Time(min) Type 101 green 10 30 Breast tissue 2 mm 102 purple 10 30 liver 2mm 103 green 10 30 liver 2 mm 104 green 20 30 liver 5 mm 105 purple 3030 Breast tissue 5 mm 106 purple 30 30 Breast tissue 5 mm 107 green 2030 liver 5 mm 108 purple 30 30 Breast tissue 4 mm 109 green 30 30 liver4 mm 110 purple 30 30 Liver 4 mm 111 purple 30 30 Breast tissue 2 mm 112purple 30 30 Breast tissue 4 mm

Control samples: Tissue specimens 2 mm to 5 mm thick were used. Thereagents and time schedule provided below is used as the controlprocedure in all examples. After processing, embedding, and sectioning;reference slides are compared with slides processed by the presentexemplary embodiment of the invention. Hematoxylin-eosin staining isused for pathological comparison.

TABLE 1.2 Control Specimen processing Schedule Time Station Reagentexposure 1  10% Buffered neutral formalin 120 min 2  10% Bufferedneutral formalin 120 min 3  70% Isopropyl alcohol 30 min. 4  80%Isopropyl alcohol 30 min. 5  95% Isopropyl alcohol 30 min. 6  95%Isopropyl 30 min. 7 100% isopropyl alcohol 45 min. 8 100% Isopropyl 45min. 9 Xylene 45 min. 10 Xylene 45 min. 11 Paraffin 60 min. 12 Paraffin60 min Embed and section

The dissolving compound of Table 1.0 is transferred from the chemicaltank to the solvent regenerator and heated for 15 minutes to anequilibrium temperature of 60° C. with a resulting pressure of 0.8 bars(12 psig). The heated compound is then transferred to the reactorcontainer holding specimens listed in Table 1.1.

The container conditions are next equilibrated to 60° C., 0.8 bars (12psig), a flow rate of 2.35 liter/min, and a space velocity of 91.2-sec.Specimens are processed for individual times as indicated in Table 1.1.After this processing, specimens are super-saturated with the compound.The excess compound is then returned to the regenerator for additionalprocessing, regeneration and solute separation.

Paraffin is next charged into the reaction container. The temperatureinside the container is stabilized to 60° C., and a chemical-potentialdifference is created between the Paraffin and compound-saturatedspecimens. Vacuum is then used to increase this differential, vaporizingthe compound within the specimen, and substituting compound moleculeswith hot Paraffin. Vacuum is continually applied until a pressureequilibration is achieved. Ten minutes after equilibrium is obtained,the heaters are disengaged, the vent solenoid opens, and the reactioncontainer is brought to room temperature before extracting thespecimens.

Results: Sections of the tissue obtained from the above processing wereexcellent and virtually indistinguishable from the reference specimensusing a light microscope. Breast tissue processed using the presentinvention did not have to be defatted prior to processing. The solventremoved all lipids that interfere with Paraffin impregnation.

Example 2

The following is an example of batch solvent blending within thereaction container. Table 2.0 shows wt-% of components before and afterbatch reactor blending.

TABLE 2.0 Solvent Batch blending Composition of Dissolving Compoundbefore and after. Wt-% Before Reactor Wt-% After Reactor ComponentBlending Blending Acetone 65.55 61.51 Hexane 16.76 15.73 DMSO  4.59 4.312-propanol 13.11 12.30 Paraffin — 6.15

A measured volume of non-Paraffin based solvent is loaded into thesolvent regenerator and heated to 60° C. Liquid Paraffin is transferredinto the sealed and heated reaction container until the Paraffin levelindicator switch is triggered. Dissolving compound is transferred fromthe solvent regenerator to the reaction container. The compound andParaffin are thoroughly blended for 5 minutes. The blend is thentransferred to the regenerator. The container is evacuated and loadedwith the specimens. The histoprocessing begins.

TABLE 2.1 Typical processing times and specimen type. ProcessingReference Color code Time Impregnation Tissue No. Code (minutes) Time(min) Type 201 green 10 30 Breast tissue 2 mm 202 purple 10 30 liver 2mm 203 green 10 30 liver 2 mm 204 green 20 30 liver 5 mm 205 purple 3030 Breast tissue 5 mm 206 purple 30 30 Breast tissue 5 mm 207 green 2030 liver 5 mm 208 purple 30 30 Breast tissue 4 mm 209 green 30 30 liver4 mm 210 purple 30 30 Liver 4 mm 211 purple 30 30 Breast tissue 2 mm 212purple 30 30 Breast tissue 4 mm

The dissolving compound of Table 2.0, in the solvent regenerator, isheated to an equilibrium temperature of 60° C. and a pressure of about0.8 bars (12 psig), then transferred to the reaction container holdingthe specimens listed in Table 2.1.

The container conditions are next equilibrated to 60° C., and about 1.6bars (25 psig), a flow rate of 2.35 liter/min, and a space velocity of91.2-sec. Specimens are processed for individual times as indicated inTable 2.1. After this processing, specimens are super-saturated with thedissolving compound. The excess compound is then returned to theregenerator for additional processing, regeneration and soluteseparation.

Paraffin is next charged to the reaction container. The container isstabilized to 60° C., and a chemical-potential difference is createdbetween the Paraffin and compound saturated specimens. Vacuum is thenused to increase this differential, vaporizing the compound with in thespecimen, and substituting compound molecules with hot Paraffin. Vacuumis continually applied until a pressure equilibration is achieved. Tenminutes after equilibrium is obtained, the heaters are disengaged, thevent solenoid opens, and the reaction container is brought to roomtemperature before extracting the specimens.

Results: The section of the tissue obtained from the above processingwas excellent and virtually indistinguishable under a light microscopefrom sample process using the control procedure.

Example 3

The following is another example of batch solvent blending within thereaction container. Table 3.0 shows wt-% of components before and afterbatch reactor blending.

TABLE 3.0 Solvent Batch blending Composition of Dissolving Compoundbefore and after. Wt-% Before Reactor Wt-% After Reactor ComponentBlending Blending Acetone 17.45 16.68 Hexane 43.16 38.80 DMSO  5.63 5.062-propanol 33.76 10.12 Paraffin — 30.35

TABLE 3.1 Typical processing times and specimen type. ProcessingReference Color code Time Impregnation Tissue No. Code (minutes) Time(min) Type 301 green 10 30 Breast tissue 2 mm 302 purple 10 30 liver 2mm 303 green 10 30 liver 2 mm 304 green 20 30 liver 5 mm 305 purple 3030 Breast tissue 5 mm 306 purple 30 30 Breast tissue 5 mm 307 green 2030 liver 5 mm 308 purple 30 30 Breast tissue 4 mm 309 green 30 30 liver10 mm 310 purple 30 30 Liver 10 mm 311 purple 30 30 Breast tissue 2 mm312 purple 30 30 Breast tissue 4 mm

The dissolving compound of Table 3.0 in the solvent regenerator isheated to an equilibrium temperature of 60° C. and a pressure of about0.8 bars (12 psig). The heated compound is then transferred to thereaction container holding the specimens listed in Table 3.1.

The container conditions are next equilibrated to 60° C., about 2.4 bars(36.5 psig, a flow rate of 2.35 liter/min, and a space velocity of91.2-sec. Specimens are processed for individual times as indicated inTable 3.1. After this processing, specimens are super-saturated with thedissolving compound. The excess compound is then returned to theregenerator.

Additional Paraffin is next charged to the container. The container isstabilized to 60° C., and a chemical-potential difference is createdbetween the Paraffin and compound-saturated specimens. Vacuum is thenused to increase this differential, vaporizing the dissolving compoundwith in the specimen, and substituting the compound molecules with hotParaffin. Vacuum is continually applied until a pressure equilibrationis achieved. Ten minutes after equilibrium is obtained, the heaters aredisengaged, the vent solenoid opens, and the reaction container isbrought to room temperature before extracting the specimens.

Results: The section of the tissue obtained from the above processingwas excellent and virtually indistinguishable under a light microscopefrom sample process using the control procedure.

Example 4

In this example, no Paraffin is added to the solvent mixture. Theimpregnation set takes an additional 20 minutes.

TABLE 4.0 Composition of Dissolving Compound Component Weight-percentAcetone 45.8 Hexane 25.4 DMSO 7.0 2-propanol 19.8

TABLE 4.1 Typical processing times and specimen type. ProcessingReference Color code Time Impregnation Tissue No. Code (minutes) Time(min) Type 401 green 10 50 Breast tissue 2 mm 402 purple 10 50 liver 2mm 403 green 10 50 liver 2 mm 404 green 20 50 liver 5 mm 405 purple 3050 Breast tissue 5 mm 406 purple 30 50 Breast tissue 5 mm 407 green 2050 liver 5 mm 408 purple 30 50 Breast tissue 4 mm 409 green 30 50 liver10 mm 410 purple 30 50 Liver 10 mm 411 purple 30 50 Breast tissue 2 mm412 purple 30 50 Breast tissue 4 mm

The dissolving compound of Table 4.0 is loaded into the solventregenerator and heated for 15 minutes to an equilibrium temperature of60° C. and a resulting pressure of about 0.8 bars (12 psig). The heatedcompound is then transferred to the reaction container holding sampleslisted in Table 4.1.

The container conditions are next equilibrated to 60° C., 0.8 bars (12psig), a flow rate of 2.35 liter/min, and a space velocity of 91.2-sec.Specimens are processed for individual times as indicated in Table 4.1.After this processing, specimens are super-saturated with the dissolvingcompound. The excess compound is then returned to the regenerator.

Paraffin is next charged to the reaction container. The container isstabilized to 60° C., and a chemical-potential difference is createdbetween the Paraffin and compound saturated specimens. Vacuum is thenused to increase this differential, vaporizing the compound with in thespecimen, and substituting compound molecules with hot Paraffin. Vacuumis continually applied until a pressure equilibration is achieved. Tenminutes after equilibrium is obtained, the heaters are disengaged, thevent solenoid opens, and the reaction container is brought to roomtemperature before extracting the specimens.

Results: The section of the tissue obtained from the above processingwas excellent and virtually indistinguishable under a light microscopefrom sample process using the control procedure.

Example 5

In this example, no Paraffin is directly added to the solvent mixture.Paraffin is used to displace the dissolving compound that has saturatedthe specimens. The impregnation set takes an additional 20 minutes.

TABLE 5.0 Composition of Dissolving Compound Component Weight-percentAcetone 59 Hexane 41 DMSO 2-propanol

TABLE 5.1 Typical processing times and specimen type. ProcessingReference Color code Time Impregnation Tissue No. Code (minutes) Time(min) Type 501 green 10 50 Breast tissue 2 mm 502 purple 10 50 liver 2mm 503 green 10 50 liver 2 mm 504 green 20 50 liver 5 mm 505 purple 3050 Breast tissue 5 mm 506 purple 30 50 Breast tissue 5 mm 507 green 2050 liver 5 mm 508 purple 30 50 Breast tissue 4 mm 509 green 30 50 liver10 mm 510 purple 30 50 Liver 10 mm 511 purple 30 50 Breast tissue 2 mm512 purple 30 50 Breast tissue 4 mm

The dissolving compound of Table 5.0 is loaded into the solventregenerator and heated for 15 minutes to an equilibrium temperature of60° C. and a resulting pressure of about 0.8 bars (12 psig). The heatedcompound is then transferred to the reaction container holding thespecimens listed in Table 5.1.

The reaction container conditions are next equilibrated to 60° C., about0.8 bars (12 psig), a flow rate of 2.35 liter/min, and a space velocityof 91.2-sec. Specimens are processed for individual times as indicatedin Table 5.1. After this processing, specimens are super-saturated withthe compound. The excess compound is then returned to the regenerator.

Paraffin is next charged into the reaction container. The container isstabilized to 60° C., and a chemical-potential difference is createdbetween the Paraffin and compound-saturated specimens. Vacuum is thenused to increase this differential, vaporizing the compound within thespecimen, and substituting compound molecules with hot Paraffin. Vacuumis continually applied until a pressure equilibration is achieved. Tenminutes after equilibrium is obtained, the heaters are disengaged, thevent solenoid opens, and the reaction container is brought to roomtemperature before extracting the specimens.

Results: The section of the tissue obtained from the above processingwas excellent and virtually indistinguishable under a light microscopefrom sample process using the control procedure.

Example 6

The following is an example of batch solvent blending within thereaction container. Table 6.0 shows wt-% of components before and afterbatch reactor blending.

TABLE 6.0 Solvent Batch blending Composition of Dissolving compoundbefore and after. Wt-% Before Reactor Wt-% After Reactor ComponentBlending Blending Acetone 59.0 54.0 Hexane 41.0 38.0 Paraffin — 8.0

TABLE 6.1 Typical processing times and specimen type. ProcessingReference Color code Time Impregnation Tissue No. Code (minutes) Time(min) Type 601 green 10 50 Breast tissue 2 mm 602 purple 10 50 liver 2mm 603 green 10 50 liver 2 mm 604 green 20 50 liver 5 mm 605 purple 3050 Breast tissue 5 mm 606 purple 30 50 Breast tissue 5 mm 607 green 2050 liver 5 mm 608 purple 30 50 Breast tissue 4 mm 609 green 30 50 liver10 mm 610 purple 30 50 Liver 10 mm 611 purple 30 50 Breast tissue 2 mm612 purple 30 50 Breast tissue 4 m

The dissolving compound residing in the solvent regenerator isconditioned by heating it to an equilibrium temperature of 60° C. and apressure of about 0.8 bars (12 psig). The heated compound is thentransferred to the reaction container holding the tissue specimens. Anyexcess compound is returned to the solvent regenerator.

The reaction container conditions are next equilibrated to 60° C., about1.6 bars (25 psig), a flow rate of 2.35 liter/min, and a space velocityof 91.2-sec. Specimens are processed for individual times as indicatedin Table 6.1. After this processing, specimens are super-saturated withthe dissolving compound.

Next, the reaction container is flooded with Paraffin. The container isrestabilized to 60° C., and a chemical-potential difference is createdbetween the Paraffin and the dissolving compound-saturated specimens.Vacuum is then used to increase this differential, vaporizing thecompound within the specimens. The compound molecules are replaced withhot Paraffin. Vacuum is continually applied until a pressureequilibration is achieved. Ten minutes after equilibrium is obtained,the heaters are disengaged, the vent solenoid opens, and the reactioncontainer is brought to room temperature before extracting thespecimens.

Results: The section of the tissue obtained from the above processingwas excellent and virtually indistinguishable under a light microscopefrom sample process using the control procedure.

Example 7

The following is a first example of batch blending ones zeotrope solventmix, and Paraffin within the reaction container. Table 7.0 shows wt-% ofcomponents before and after batch reactor blending.

TABLE 7.0 Zeotrope Solvent blended before and after Paraffin addition.Wt-% Before Reactor Wt-% After Reactor Component Blending BlendingAcetone 53.0 50.5 Xylenes 26.0 24.8 Ethanol 20.0 19.0 DMSO  1.0 1.0Paraffin — 4.7

Outline of Batch Solvent Blending:

3.8 liters (1 gallon) of non-Paraffin based solvent is placed into thechemical tank 29 and is transferred to the solvent regenerator 28 forheating to 60° C.

The reaction container 11 is sealed, the jacket heater 65 is energized,the vacuum pump 31 is engaged, the control valve 42 is opened and liquidParaffin is transferred from the Paraffin make-up vessel 27 to thereaction container until the Paraffin level indicator is energized. Atthis time, the control valve is closed, and the vacuum pump is stopped.Control valves 43, 44, 45 are opened, so that the solvent is transferredfrom the solvent regenerator to the reaction container. When the highlevel indicator switch 68 is energized, solvent pump 30 is stopped, andall control valves are positioned to create a reaction container loopconfiguration. The heat exchanger 60 and solvent pump 30 are energized,and the solvent is blended for ten minutes. The blend loop isreconfigured, so that the blended solvent is then transferred to thesolvent regenerator 28. The reaction container is evacuated. The systemis now ready to process specimens.

TABLE 7.1 Typical processing times and specimen type. ProcessingReference Color code Time Impregnation Tissue No. Code (minutes) Time(min) Type 701 green 10 30 Breast tissue 2 mm 702 purple 10 30 liver 2mm 703 green 10 30 liver 2 mm 704 green 20 30 liver 5 mm 705 purple 3030 Breast tissue 5 mm 706 purple 30 30 Breast tissue 5 mm 707 green 2030 liver 5 mm 708 purple 30 30 Breast tissue 4 mm 709 green 30 30 liver10 mm 710 purple 30 30 Liver 10 mm 711 purple 30 30 Breast tissue 2 mm712 purple 30 30 Breast tissue 4 mm

Tissue Processing: Extraction solvent (Solvent) in Table 7.0, in thesolvent regenerator, is heated to an equilibrium temperature of 60° C.and a pressure of 0.8 bars (12 psig). The heated solvent is thentransferred to the reaction container containing samples listed in Table7.1.

The reaction container conditions are next equilibrated to 60° C., 1.6bars (25 psig), a flow rate of 2.35 liters/min, and a space velocity of91.2-sec. Specimens are processed for individual times as indicated inTable 7.1. After this processing, the specimens are super-saturated withthe solvent. The excess reaction container solvent is then returned tothe solvent regenerator for additional processing, solvent regenerationand solute separation.

Paraffin is next charged to the reaction container via Paraffin makeupvessel. The reaction container is stabilized to 60° C., and achemical-potential difference is created between the Paraffin and thesolvent-saturated specimens. Vacuum is then used to increase thisdifferential, vaporizing the solvent within the specimen, andsubstituting solvent molecules with hot Paraffin. Vacuum is continuallyapplied until a pressure equilibration is achieved. Ten minutes afterequilibrium is obtained, the heaters are disengaged, the vent solenoidopens, and the reaction container is brought to room temperature beforeextracting the specimens.

Results: The section of the tissue obtained from the above processingwas excellent and virtually indistinguishable under a light microscopefrom sample process using the control procedure.

Example 8

The following is an example of batch blending of a zeotropic solvent andParaffin within the reaction container. Table 8.0 shows wt-% ofcomponents before and after batch reactor blending.

TABLE 8.0 Zeotrope Solvent blended before and after Paraffin addition.Wt-% Before Reactor Wt-% After Reactor Component Blending BlendingAcetone 62.0 58.5 Xylenes 19.0 18.0 Ethanol 19.0 17.9 Paraffin — 5.6

Outline of Batch Solvent Blending:

3.8 liters (1 gallon) of non-Paraffin based solvent is placed into thechemical tank and is transferred to the Solvent regenerator for heatingto 60° C.

The reaction container is sealed, the jacket heater is energized, thevacuum pump is engaged, the control valve 42 is opened and liquidParaffin is transferred from the Paraffin makeup vessel to the reactioncontainer until the Paraffin level switch is energized. At this time,the control valve is closed, and the vacuum pump is stopped. Controlvalves 43,44,45 are opened, so that the solvent is transferred from thesolvent regenerator to the reaction container. When the high levelindicator switch is energized, the solvent pump is stopped, and allcontrol valves are positioned to create a reaction container loopconfiguration. The heat exchanger and solvent pump are energized, andthe solvent is blended for ten minutes. The blend loop is reconfigured,so that the blended solvent is then transferred to the solventregenerator. The reaction container is evacuated. The system is nowready to process specimens.

TABLE 8.1 Typical processing times and specimen type. ProcessingReference Color code Time Impregnation Tissue No. Code (minutes) Time(min) Type 801 green 10 30 Breast tissue 2 mm 802 purple 10 30 liver 2mm 803 green 10 30 liver 2 mm 804 green 20 30 liver 5 mm 805 purple 3030 Breast tissue 5 mm 806 purple 30 30 Breast tissue 5 mm 807 green 2030 liver 5 mm 808 purple 30 30 Breast tissue 4 mm 809 green 30 30 liver10 mm 810 purple 30 30 Liver 10 mm 811 purple 30 30 Breast tissue 2 mm812 purple 30 30 Breast tissue 4 mm

Tissue Processing Extraction solvent (Solvent) in Table 8.0, in thesolvent regenerator, is heated to an equilibrium temperature of 60° C.and a pressure of 0.8 bars (12 psig). The heated solvent is thentransferred to the reaction container containing samples listed in table8.1.

The reaction container conditions are next equilibrated to 60° C., 1.6bars (25 psig), a flow rate of 2.35 liters/min, and a space velocity of91.2-sec. Specimens are processed for individual times as indicated inTable 8.1. After this processing, the specimens are super-saturated withthe solvent. The excess reaction container solvent is then returned tothe solvent regenerator for additional processing, solvent regenerationand solute separation.

Paraffin is next charged to the reaction container via Paraffin makeupvessel. The reaction container is stabilized to 60° C., and achemical-potential difference is created between the Paraffin andsolvent-saturated specimens. Vacuum is then used to increase thisdifferential, vaporizing the solvent within the specimen, andsubstituting solvent molecules with hot Paraffin. Vacuum is continuallyapplied until a pressure equilibration is achieved. Ten minutes afterequilibrium is obtained, the heaters are disengaged, the vent solenoidopens, and the reaction container is brought to room temperature beforeextracting the specimens.

Results: The section of the tissue obtained from the above processingwas excellent and virtually indistinguishable under a light microscopefrom sample process using the control procedure.

Other tissue types can be processed using the present inventionembodiments, such as but not limited to: appendix, bowel, fallopian tub,kidney, liver, lung, parotid, placenta, prostate, thyroid, adenoma,cervix, skin and many others.

While the preferred embodiments of the invention has been described,modifications can be made and other embodiments may be devised withoutdeparting from the spirit of the invention and the scope of the appendedclaims.

1. A tissue processing method which comprises: providing a pressure andtemperature controllable reaction container; placing at least one freshtissue specimen in said container; treating said specimen in saidcontainer to remove solutes and replace said solutes with Paraffin, saidstep of treating consisting essentially of: introducing a temperatureand pressure conditioned dissolving compound into said container;allowing time for said compound to penetrate said specimen and to dilutesolutes therein; flooding said container with liquid Paraffin;vaporizing said compound and allowing a period for said Paraffin toimpregnate said specimens; evacuating said compound and diluted solutesfrom said container; and reducing the temperature in said containerbelow the melting point of Paraffin.
 2. The method of claim 1, whereinsaid dissolving compound comprises a mixture taken from the groupconsisting of azeotropic and zeotropic dehydrant mixtures.
 3. The methodof claim 1, wherein said dissolving compound comprises a solvent takenfrom a group consisting essentially of ketones, esters, alcohols,aldehydes, ethers, aromatics, low molecular weight hydrocarbons, and amixture of low molecular weight hydrocarbons.
 4. The method of claim 1,wherein said dissolving compound comprises a mixture taken from a groupconsisting essentially of: a mixture of about 60% per weight Acetone andabout 40% per weight of at least one Hexane; a mixture of about 46% perweight Acetone, about 25% per weight of at least one Hexane, about 7%per weight DMSO, about 20% per weight 2-Propanol and about 2% per weightParaffin; a mixture of about 62% per weight Acetone, about 16% perweight of at least one Hexane, about 4% per weight DMSO, about 12% perweight 2-Propanol and about 6% per weight Paraffin; a mixture of about17% per weight Acetone, about 39% per weight of at least one Hexane,about 5% per weight DMSO, about 12% per weight 2-Propanol and about 30%per weight Paraffin; a mixture of about 46% per weight Acetone, about25% per weight of at least one Hexane, about 7% per weight DMSO andabout 20% per weight 2-Propanol; a mixture of about 53% per weightAcetone, about 26% per weight at least one Xylene, about 20% per weightEthanol, and about 1% per weight DMSO; and, a mixture of about 62% perweight Acetone, about 19% per weight at least one Xylene, and about 19%per weight Ethanol.
 5. The method of claim 1, wherein said dissolvingcompound consists of a mixture taken from a group consisting essentiallyof: a mixture of about 60% per weight Acetone and about 40% per weightof at least one Hexane; a mixture of about 46% per weight Acetone, about25% per weight of at least one Hexane, about 7% per weight DMSO, about20% per weight 2-Propanol and about 2% per weight Paraffin; a mixture ofabout 62% per weight Acetone, about 16% per weight of at least oneHexane, about 4% per weight DMSO, about 12% per weight 2-Propanol andabout 6% per weight Paraffin; a mixture of about 17% per weight Acetone,about 39% per weight of at least one Hexane, about 5% per weight DMSO,about 12% per weight 2-Propanol and about 30% per weight Paraffin; amixture of about 46% per weight Acetone, about 25% per weight of atleast one Hexane, about 7% per weight DMSO and about 20% per weight2-Propanol; a mixture of about 53% per weight Acetone, about 26% perweight at least one Xylene, about 20% per weight Ethanol, and about 1%per weight DMSO; and, a mixture of about 62% per weight Acetone, about19% per weight at least one Xylene, and about 19% per weight Ethanol. 6.The method of claim 1, which further comprises; regenerating an amountof said dissolving compound evacuated from said container in aregeneration canister.
 7. The method of claim 6, wherein saidregenerating comprises: distilling said evacuated compound; andconverting vent waste gases into carbon dioxide and water through athermocatalytic oxidizer.
 8. The method of claim 6, which furthercomprises conditioning regenerated dissolving compound for introductioninto said reaction container.
 9. The method of claim 8, wherein saidconditioning comprises: heating said dissolving compound in a solventvessel to a temperature of approximately 60° C.; and establishing apressure of approximately 0.8 bars (12 psig) in said vessel.
 10. Themethod of claim 1, wherein said time is limited to no more thanapproximately 30 minutes, and said period is limited to no more thanapproximately 30 minutes.
 11. The method of claim 10, wherein saidspecimens are taken from a group consisting essentially of non-processedand non-burred tissue sections up to 5 mm thick.
 12. The method of claim1, wherein: said time is limited to no more than approximately 30minutes, and said period is limited to no more than approximately 50minutes; and said specimens comprise non-processed and non-burred tissuesections up to 10 mm thick.
 13. The method of claim 1, which furthercomprises admitting a limited amount of Paraffin into said containerbefore introducing said dissolving compound.
 14. The method of claim 1,wherein said dissolving compound has a refractive index between about1.350 and 1.499 at 20° C.
 15. The method of claim 1, wherein saiddissolving compound comprises a lubricant for process pumps and valves.16. The method of claim 1, wherein said dissolving compound comprises amixture of approximately 17% to 65% per weight Acetone and 16% to 40%per weight at least one Hexane.
 17. The method of claim 1, wherein saiddissolving compound is vaporizable at a pressure of no more than 10 torrand a temperature of no more than about 80° C.
 18. The method of claim17, wherein said dissolving compound has a refractive index betweenabout 1.350 and 1.499 at 20° C.
 19. A histoprocess which comprises:dissolving and removing cellular solutes in a tissue specimen using asingle dissolving compound; and replacing said solutes with Paraffin;said process being performed in a continuous sequence of steps within asingle vessel.
 20. The histoprocess of claim 19, wherein said dissolvingcompound comprises a mixture taken from the group consisting ofazeotropic and zeotropic dehydrant mixtures.
 21. The histoprocess ofclaim 19, wherein said dissolving compound comprises a solvent takenfrom a group consisting essentially of ketones, esters, alcohols,aldehydes, ethers, aromatics, low molecular weight hydrocarbons, and amixture of low molecular weight hydrocarbons.
 22. The histoprocess ofclaim 19, wherein said dissolving compound comprises Acetone and atleast one Hexane.
 23. The histoprocess of claim 19, wherein said stepscomprise: saturating said specimen with said dissolving compound;flooding said vessel with liquid Paraffin; and vaporating saiddissolving compound and said solutes to allow said Paraffin toinfiltrate spaces vacated by said compound and solutes.
 24. Thehistoprocess of claim 19, which further comprises: evacuating saiddissolving compound and solutes to a regeneration canister; recuperatingsaid dissolving compound by distillation; and disposing of vent gases bythermocatalytic oxidation.
 25. The histoprocess of claim 19, whichfurther comprises mixing said compound in said vessel prior to saiddissolving and removing.
 26. The histoprocess of claim 19, wherein saiddissolving compound comprises a lubricant for process pumps and valves.27. The histoprocess of claim 19, wherein said dissolving compound has arefractive index between about 1.350 and 1.499 at 20° C.
 28. Thehistoprocess of claim 19, wherein said dissolving compound comprises amixture of approximately 17% to 65% per weight Acetone and 16% to 40%per weight at least one Hexane.
 29. The histoprocess of claim 19,wherein said dissolving compound is vaporizable at a pressure of no morethan 10 torr and a temperature of no more than about 80° C.
 30. Thehistoprocess of claim 19, wherein: said time is limited to no more thanapproximately 30 minutes, and said period is limited to no more thanapproximately 50 minutes; and said specimens comprise non-processed andnon-burred tissue sections up to 10 mm thick.
 31. The histoprocess ofclaim 19, which further comprises admitting a limited amount of Paraffininto said container before introducing said dissolving compound.
 32. Anapparatus for processing tissue specimens against decomposition,putrefaction and autolysis which comprises: a hermetically sealablereaction container; means for controlling the temperature and pressureinside said container; a source of dissolving compound connectable tosaid container; a source of melted Paraffin connectable to saidcontainer; and means for evacuating gases from said container; whereinsaid dissolving compound comprises a mixture of a ketone and at leastone low molecular weight hydrocarbon.
 33. The apparatus of claim 32,wherein said dissolving compound comprises a mixture taken from thegroup consisting of azeotropic and zeotropic dehydrant mixtures.
 34. Theapparatus of claim 32, wherein said dissolving compound comprises amixture taken from a group consisting essentially of: a mixture of about60% per weight Acetone and about 40% per weight at least one Hexane; amixture of about 46% per weight Acetone, about 25% per weight at leastone Hexane, about 7% per weight DMSO, about 20% per weight 2-Propanoland about 2% per weight Paraffin; a mixture of about 62% per weightAcetone, about 16% per weight at least one Hexane, about 4% per weightDMSO, about 12% per weight 2-Propanol and about 6% per weight Paraffin;a mixture of about 17% per weight Acetone, about 39% per weight at leastone Hexane, about 5% per weight DMSO, about 12% per weight 2-Propanoland about 30% per weight Paraffin; a mixture of about 46% per weightAcetone, about 25% per weight at least one Hexane, about 7% per weightDMSO and about 20% per weight 2-Propano; a mixture of about 53% perweight Acetone, about 26% per weight at least one Xylene, about 20% perweight Ethanol, and about 1% per weight DMSO; and, a mixture of about62% per weight Acetone, about 19% per weight at least one Xylene, andabout 19% per weight Ethanol.
 35. The apparatus of claim 32, whereinsaid dissolving compound consists of a mixture taken from a groupconsisting essentially of: a mixture of about 60% per weight Acetone andabout 40% per weight at least one Hexane; a mixture of about 46% perweight Acetone, about 25% per weight at least one Hexane, about 7% perweight DMSO, about 20% per weight 2-Propanol and about 2% per weightParaffin; a mixture of about 62% per weight Acetone, about 16% perweight at least one Hexane, about 4% per weight DMSO, about 12% perweight 2-Propanol and about 6% per weight Paraffin; a mixture of about17% per weight Acetone, about 39% per weight at least one Hexane, about5% per weight DMSO, about 12% per weight 2-Propanol and about 30% perweight Paraffin; a mixture of about 46% per weight Acetone, about 25%per weight at least one Hexane, about 7% per weight DMSO and about 20%per weight 2-Propano; a mixture of about 53% per weight Acetone, about26% per weight at least one Xylene, about 20% per weight Ethanol, andabout 1% per weight DMSO; and, a mixture of about 62% per weightAcetone, about 19% per weight at least one Xylene, and about 19% perweight Ethanol.
 36. The apparatus of claim 32, wherein Paraffin presentin the solvent provides lubricating properties for process pumps andvalves.
 37. The apparatus of claim 32, wherein said dissolving compoundcomprises a solvent taken from the group consisting essentially ofketones, esters, alcohols, aldehydes, ethers, aromatics, low molecularweight hydrocarbons, and a mixture of low molecular weight hydrocarbons.38. The apparatus of claim 32, which further comprises means forregenerating vaporized dissolving compound evacuated from saidcontainer.
 39. The apparatus of claim 38, wherein said means forregenerating comprise: a still for recuperating said evacuateddissolving compound; and a thermocatalytic oxidizer for converting ventwaste gases into carbon dioxide and water.
 40. The apparatus of claim32, wherein said dissolving compound has a refractive index betweenabout 1.350 and 1.499 at 20° C.
 41. The apparatus of claim 32, whereinsaid dissolving compound comprises a mixture of approximately 17% to 65%per weight Acetone and 16% to 40% per weight at least one Hexane. 42.The apparatus of claim 32, wherein: said time is limited to no more thanapproximately 30 minutes, and said period is limited to no more thanapproximately 50 minutes; and said specimens comprise non-processed andnon-burred tissue sections up to 10 mm thick.
 43. The combination of atissue specimen with a preparation for dissolving lipids and othercellular solutes in said specimen, said preparation being taken from agroup consisting essentially of: a mixture of about 60% per weightAcetone and about 40% per weight at least one Hexane; a mixture of about46% per weight Acetone, about 25% per weight at least one Hexane, about7% per weight DMSO, about 20% per weight 2-Propanol and about 2% perweight Paraffin; a mixture of about 62% per weight Acetone, about 16%per weight at least one Hexane, about 4% per weight DMSO, about 12% perweight 2-Propanol and about 6% per weight Paraffin; a mixture of about17% per weight Acetone, about 39% per weight at least one Hexane, about5% per weight DMSO, about 12% per weight 2-Propanol and about 30% perweight Paraffin; a mixture of about 46% per weight Acetone, about 25%per weight at least one Hexane, about 7% per weight DMSO and about 20%per weight 2-Propanol; a mixture of about 53% per weight Acetone, about26% per weight at least one Xylene, about 20% per weight Ethanol, andabout 1% per weight DMSO; and, a mixture of about 62% per weightAcetone, about 19% per weight at least one Xylene, and about 19% perweight Ethanol.
 44. A process for removing cellular solutes from atissue specimen which comprises treating said specimen with a dissolvingcompound having a viscosity of no more than about 1.30 centipoises at60° C. and a boiling point of more than about 80° C. at a pressure of nomore than 10 torr.
 45. The process of claim 44, wherein said compound iszeotropic.
 46. The process of claim 44, wherein said dissolving compoundhas a viscosity of no more than about 0.75 centipoises at 60° C. and aboiling point of less than about 80° C. at a pressure of no more than 10torr.
 47. The process of claim 44, wherein said compound is regeneratedafter combination with said solutes.
 48. The process of claim 44,wherein said dissolving compound is azeotropic.
 49. The process of claim44, wherein said dissolving compound comprises a solvent taken from agroup consisting essentially of ketones, esters, alcohols, aldehydes,ethers, aromatics, low molecular weight hydrocarbons, and a mixture oflow molecular weight hydrocarbons.
 50. The process of claim 44, whereinsaid dissolving compound comprises a lubricant for process pumps andvalves.